Cell-Free Expression of Membrane Proteins with Applications to Drug Discovery

The goal of this project is to demonstrate the ability to express functionally-active, membrane- bound proteins in a cell-free transcription-translation (TX-TL) system that has been developed at Caltech. We will make use of a novel nanodisc structure that emulates the cell membrane and has been successfully tested at Amgen. The TX-TL breadboard has been demonstrated to enable rap- id, cost-effective, and high-throughput exploration of complex biomolecular circuits (Niederholtmeyer et al. 2015), metabolic engineering pathways (Wu et al. 2015), and engineered enzyme variants (de los Santos et al. 2015). We believe that given these three applications, the TX-TL breadboard can also enable expression and assay of functionally-active, membrane-bound proteins. Building on a recently established collaboration between Caltech and Amgen, we propose to demonstrate the utility of this approach for automation-enabled drug discovery.

Current participants: Additional participants:

Collaborators: Ching Chen (Amgen) Past participants: Abel Chiao* Zach Sun

Objectives

High-level cell expression of membrane proteins is often difficult or self-prohibited due to cell toxicity. Purification and reconstitution of membrane-bound proteins has also proven to be very challenging compared to non-membrane bound analogues. The direct cell-free expression of challenging membrane-bound proteins provides an attractive alternative to overcome these difficulties. This project aims to achieve high-level expression and display of membrane proteins by integration of two technologies: (1), cell-free expression, and (2), assembly of membrane proteins into nanodiscs. The successful implementation of the combined technologies will produce and display membrane proteins in nanodiscs with defined size and lipid components. Together, it will enable us to develop robust and reliable measurements of kinetic and equilibrium binding for membrane proteins.

References

• On-chip membrane protein cell-free expression enables development of a direct binding assay: A curious case of potassium channel KcsA-Kv1.3. A. Vaisha, S. Guo, R. M.Murray, P. J..Grandsard and Q. Chen. Analytical Biochemistry, 556:70-77, 2018.

Research supported through an Amgen Chem-Bio-Engineering Award (CBEA).

Agency: Amgen Grant number: AMGEN.CBEARMM

Start date: 1 Nov 2015 End date: 31 Dec 2016

Support: ~1 graduate student, technician support + supplies Reporting: Presentation at semi-annual research review + short written summary of research (annually).